Apparatus for fractionating colloid dispersions



S. RAYMOND Aug. 14, 1956 APPARATUS FOR FRACTIONATING COLLOID DISPERSIONS Filed July 22. 1952 3 Sheets-Sheet 1 INVENTOR:

S. RAYMOND Aug. 14, 1956 APPARATUS FOR FRACTIONATING COLLOID DISPERSIONS Filed July 22, 1952 3 Sheets-Sheet 3 w? W/ my m y 11 a] r o w 7////// /M%M W 3 FL f FIG.

I INVENTOR;

United States Patent APPARATUS FOR FRACTIONATIN G COLLOID DISPERSIONS Samuel Raymond, New York, N. Y.

Application July 22, 1952, Serial No. 300,246

6 Claims. (Cl. 204-301) This invention relates to apparatus for effecting electrophoresis-convection, which is amethod for fractionating or separating a mixture of colloids into its components. The method operates by creating, in a dispersion of such a mixture, a horizontal electric field, which causes a horizontal migration of the colloid par ticles. The horizontal migration proceeds as far as certain vertical barriers of semipermeable membrane, where the migration is stopped and the particles accumulate, causing differences of density in the dispersion near the membranes. These differences in density cause vertical convection currents to flow in the dispersion, transporting the distinct components at different rates, thus effecting their separation. The process as a whole proceeds within a cell, whereby the separated components accumulate in different parts of the cell, usually in reservoirs.

The fundamental principles of electrophoresis-convection (also known as electrodecantation or electrostratification) have been known since at least 1924 (see, for example, the discussion in Advances in Protein Chemistry, edited by M. L. Anson and John T. Edsall, vol. IV pp. 272-277: Academic Press, Inc., New York 1948. For later work in this field see Cann, Brown, and Kirkwood, J. Biol. Chem. 181, p. 164 (1949)).

The essential components of the electrophoresis-convection apparatus are: a box or container filled with buffer solution in which the other parts of the apparatus are immersed; electrodes; and between the electrodes a cell assembly with semi-permeable faces thru which the electric field passes, the said faces forming the external walls of the convection channel so defined.

Prior to my invention leakage frequently occurred from the cell. This leakage produces a great loss of efficiency by allowing the colloid dispersion under treatment to leak into the electrode compartments. An object of the present invention is to eliminate this loss of efficiency by preventing the leakage.

Prior to my invention the efficiency of the apparatus was limited by the fact that there was only a single channel for the convective current. I have found that by dividing the convective current between a plurality of vertical laminations in the channel a great increase in the efficiency of the apparatus can be obtained. This part of my invention consists not only of inventing a type of construction for such a channel but also of discovering that a laminar channel increases the efficiency of the electrophoresis-convection apparatus.

Prior to my invention the practice of electrophoresisconvection was limited to the use of no more than about ten milliamperes of current for each square centimeter of the channel face area. I have found that substantially higher currents may be used, up to at least 100 milliamperes per square centimeter, with the result that the speed of separation is greatly increased. This speed of increased separation greatly extends the usefulness of the apparatus. In particular, liable materials such as enzymes or anti-viral substances which could not be "ice processed by this method prior to my invention, cannow be separated.

Prior to my invention adequate means for cooling the cell have not been provided. In accordance with known electrical principles, passage of electric current thru the cell results. in the production of heat in the cell. This heat is especially important when using higher currents as taught in the previous paragraph of the present invention. The heat generated interferes with the proper operation of the cell by causing undesirable convection currents. It also has adverse effects on colloid dispersions in the cell, decreasing their stability. An object of the present invention is to provide a means for adequately removing the heat generated.

Prior to my invention the method of electrophoresis.- convection has involved the withdrawal of a plurality of fractions from the cell at the conclusion of a run. Nevertheless, no means has heretofore been provided for preventing the remixing of the separated fractions Within the apparatus during the time required to withdraw one or more of them from the cell. My invention provides means by which this remixing can be prevented.

Prior to my invention experts in this field have gelr erally believed that the length of time necessary to achieve any particular separation was inversely proportional to the area of the channel face, other factors being equal. That is, the larger the channel face the shorter the time required to effect a given separation. I have discovered that the time required is in fact inversely proportional to the total electric energy passed thru the cell. This discovery means that it is not necessary to use a larger channel when a larger volume of solution is under treatment, as a smaller channel will do the same work. I have used this discovery in the design of the preferred form of my invention, thus increasing materially the efficiency of the apparatus;

Prior to my invention no account was taken of the necessity for designing the channel or channels and the reservoirs if used in such a way as to eliminate turbulence in the convective flow. This turbulence greatly reduces the efficiency of the apparatus. My invention reduces this turbulence by eliminating projections and sharp corners in the cell.

Another object of this invention is to provide an apparatus for effecting electrophoresis-convection that is economical to manufacture and simple to operate, that is highly efficient in operation, and that requires a minimum of attention and maintenance.

Another object is to provide an apparatus of the type in which different colloid dispersions can be treated successively while eliminating the possibility of contamination of a dispersion by residual traces of a preceding dispersion previously treated in the same apparatus. Another object is to provide an apparatus in which a plurality of different samples of colloid dispersions may be treated separately and simultaneously, thus insuring identity of the conditions of treatment for all samples so treated.

Still another object is to provide simple and convenient means for changing the volume capacity of the cell so as to accommodate different volumes of the colloid dispersion to be treated.

In the drawings:

Fig. 1 represents an electrophoresis-convection apparatus, partly in section.

Fig. 2 represents diagrammatically a plate of the cell frame showing the position of the bag with respect to the plate.

Figs. 3 and 6 represent cross-sectional views of devices for holding the edges of the bag.

Figs. 4 and 5 show types of bags.

Fig. 7 shows diagrammatically in section thebufier 3 jar and cell frame with buffer circulating and cooling means attached.

Fig. 8 represents an electrophoresis-convection apparatus in section.

In accordance with the invention, the apparatus includes a bag capable of retaining the colloid dispersion, which bag is constructed in a leakproof manner of fiexible semipermeable membrane. A suitable method of constructing such a bag is with the use of commercially available cellophane tubing, such as Visking sausage casing. A length of such tubing, with one end sealed off, as by tying a knot in it, forms a suitable bag. .An ofl1er method of forming the required bag is shown in Fig. 4, where the lower end 51 of the tubing 56 is passed thru the tapered opening 52 in a solid member 53 and the lumen of the tubing is occluded by a plug 54 tapered to fit the opening. In this type of construction the Wall of the tubing is between the plug and the solid member. This type of construction permits the introduction of an inlet and valve 55 into the lower end of the tubing. Another, and simpler, method of construction is shown in Fig. which shows a double length of the same type of tubing already menu'oned, held in the shape of a U with both open ends at the top. In this way, the tubing is capable of retaining the liquid placed within it, and the objects of the invention are perfectly satisfied insofar as the bag is concerned. It will be evident to those skilled in the art that other suitable types of bag construction may be employed within the teaching of the present specification.

The invention also includes means for holding the bag whereby at least a portion of the bag is in the shape of a substantially fiat vertical channel, and means for exposing at least a portion of the channel to buffer external to the bag. The flexible bag as described above assumes, when filled with liquid, a shape which is typically cylindrical and is the resultant of hydrostatic forces acting on the bag. Such a shape is not suitable for the effecting of electrophoresis-comectionwithin the bag. If however at least a portion of the bag is constrained to assume the shape of a substantially fiat channel, electrophoresis-convection may be effected therein. The assembly of the bag and the means for holding it in the proper shape, which assembly is called the cell assembly, is of course used in conjunction with means for creating a horizontal electric field immersed in a suitable buffer according to the known principles of the art.

The average thickness of the channel (i. e. the horizontal dimension thereof parallel to the electric field), or of each lamina if a laminar channel is used as specified hereinafter may be from /2 to 5 mm., the preferred range being 1 to 2 mm. There are certain advantages, such as greater capacity in the channel, to using channels of greater thickness, for example from 5 to 25 mm. In the thicker channels it is advantageous to pack the channel with a material, such as glass wool or the like, which is inert and does not conduct electricity, to provide many continuous small ducts or passages in which electrophoresis-convection may take place.

The width of the channel may be substantially greater than the thickness, and the height substantially greater than the width. The optimum area of the channel face (i. e. the surface of the channel perpendicular to the electric field) depends on the total electric energy which may be passed thru the cell without excessive temperature rise, and thisin turn depends on the cooling means available, as explained hereinafter. The area may be chosen so that the current density thru the cell is from to 100 milliamperes per square centimeter. For example, in a cell where the cooling means is equivalent to 60 watts of energy, the preferred dimensions of the channel are 1 cm. wide by cm. high.

In order to fractionate in one operation a greater volume of the dispersion than can be efficiently contained in a single channel it is advantageous to have reservoirs at the top and bottom of the channel to hold the excess dispersion Where the dispersion may circulate into the channel by convection as the fractionation proceeds.

The invention also includes means for preventing the remixing of the separated fractions within the cell during the time required to withdraw one or more of them at the conclusion of the run. The means envisaged by the present invention is a device for occluding the lumen of the channel, and a specific description of one such device will be given in connection with the later description of one embodiment of the invention.

The invention also includes means for continuously circulating cooled buffer thru the electrode compartments and in contact with the cell assembly, as shown in Fig. 7. The pump 71 circulates the buffer and the heat exchanger '72 removes heat therefrom.

The foregoing and other aims and advantages of the invention will be in part apparent and in part pointed out in the following description of one embodiment as shown in Fig. l, which is a vertical partial sectional perspective view, the partial section being taken medially and perpendicular to the channel face. The cell 10 (i. e. the part of the apparatus which contains the colloid dispersion to be treated) is constructed entirely of cellophane sausage casing, and is held in shape by a frame 11, external to the cell. The bag 10 and frame 11 together form the cell assembly. For convenience the cell assembly is separable from the other parts of the apparatus, the electrodes 12 and the buffer box 13.

The electrodes may be attached by rigid supports 14 to the box 13. The preferred form of the box is a cylinder 15 mounted on a base 16. The electrodes are both perpendicular to one and the same diameter of the box, and spaced so as to be close to but not touching the plates 23, 24 of the cell frame. The spacing between each electrode and the nearest channel face may be no more than is necessary to allow circulation of buffer be tween the electrode and the channel face. A greater distance results in inefliciency in the apparatus due to excessive heat developed in the bulfer by passage of the electric current.

The cell assembly rests on the base of the box. The longest horizontal dimension of the cell assembly is made nearly equal to the inside diameter of the cylinder. The cell assembly slides into the cylinder vertically and is held in position in the box by contact of the vertical edges 21 of the cell assembly with the curved interior surface 22 of the cylinder. The cell assembly divides the box into two compartments 18, 19, one for each electrode. Buffer is pumped into one compartment thru the inlet 25 and flows thru passageways 20 shown in Fig. 2 to the other compartment; it flows out thru the outlet 26 and is kept in circulation by the pump 71 shown in Fig. 7. It is cooled by the heat exchanger 72.

The frame shown in Fig. l is constructed of inert nonelectrically-conducting plastic and consists of two plates 23, 24 and means for clamping them together shown in Fig. 3. One of the plates is shown in Fig. 2, which is a view from the internal side of the plate (i. e. the side in contact with the bag which forms the cell). The upper and lower reservoirs of the cell fit into the recesses 35 and '36 respectively. The channel connecting the two reservoirs is exposed to the buffer in which the assembly is immersed thru the channel opening 34, which may be a single opening as shown or may be divided into more than one opening, provided that a substantial portogether. This construction is repeated on each side of the cellas required to clamp the plates together with suflicient tightness. Usually four such clamps are required; The edge 41 of the bag is firmly held by the pressure of'the two plates against it; and in case a laminating sheet 42 is used the edge thereof is also held in the same manner; In larger models of this apparatus the tension which can be exerted by the simple pressure clamp shown in Fig. 3 is not sufficient to hold the. bag tightly enough. I therefore provide a positive type of clamping device for the edge of the bag shown in Fig. 6. This device is easily adapted to multilaminar channels and Fig. 6 shows how it may be adapted to a channel of three laminations, 61, 62', and 63', formed by the walls 17 of the bag and the walls 64 of a tube of semipermeable membrane within the. bag. The wall of the bag passes around the outside of a vertical hollow member 66 between the said member and grooves 67 in the cell frame, and is clamped thereby. The wall of the internal tube passes thru a vertcial slot 68 in the member 66 and passes around another vertical member 69 and is clamped thereby. By making the innermost member hollow and with a vertical slot this type of construction may be repeated to form additional laminations if desired.

When the plates are clamped together with the device shown in Fig. 3 the only exposed metal in the clamping device is the head of the screw. Prior to my invention the use of metal screws had been tried but had not been successful because of the difficulty ,in insulating the metal from the electrolytic action of the buffer. It was also generally believed that metal bolts have an adverse effect in distorting the electric field in the cell. I have found that with the type of assembly shown in Fig. 3, during the operation of the cell when the electric field is applied, the clamping device receives an electric charge corresponding to the potential in the buffer at the head of the screw, but since the body of the screw and the nut are completely insulated from the buffer, no current can flow thru the clamping device and there i therefore neither electrolytic action nor distortion of the field. Other suitable means for clamping together the parts of this cell assembly may be employed within the teaching of the present specification.

In assembling the cell assembly with the use of the frame described above the bag which forms the cell is placed on the inner surface of one face plate in the position shown by the dotted line in Fig. 2. The opening of the bag is placed at the top and preferably extends beyond the top edge of the plate as shown. The other plate is then positioned with the channel openings in register with each other and the assembly is clamped together. The lower end 44 of the bag may be placed near the lower edge or near the upper edge of the recess 36 accordingly as a larger or smaller volume capacity is desired in the lower reservoir.

When the bag in this assembly is filled with a liquid, such as the colloid dispersion to be treated, the hydrostatic pressure of the liquid causes the flattened bag to bulge out at the top and the bottom, limited by the reservoir recesses, forming the reservoirs. In the area of the channel, however, the lumen of the bag is constrained to assume a slightly curved cross-section, as shown in 27 in Fig. 1. The thickness of the channel formed in this way may vary from zero at the edges, where the bag is clamped between the plates, to about 1 to 5 mm. or even more in the center, owing to the bulge of the flexible membrane which forms the wall thereof, with no practical effect on the process of electro phoresis-convection as carried out with this apparatus. In other words, it is not necessary for the outer walls 17 of the channel 27 to be absolutely plane and parallel to each other, as a departure of 5 mm. or more from planarity does not interfere with the process of electrophoresis-convection. To prevent greater bulging than this it is necessary to restrain the wall, of the bag, somewhat. This may be done. by applying a horizontal tension to the channel wall 17'. This is done in the design shown by clamping the edges 41 of the bag securely between the faces of the plates the object being to hold the edges of the channel between the two plates against the hydrostatic forces in the channel which tend to pull it away from its proper position. The present specification teaches that it is preferable to do this While maintaining the wall of the bag intact, thus preventing the bag from leaking. The tension may be applied in other ways, for example by means of a rod within the. bag which is held by grooves in the face of each plate, as shown in Fig. 6. Other methods of holding the edges of the bag may be used, such as ribs and grooves in the surface of the plates.

Other means besides the application of tension may be used for controlling bulging of the exposed area of the channel membrane wall, such as a stiff screen or other stiff foraminiferous member pressing against the bulge. In order to make an operable electrophoresis-convection channel this member must have two characteristics, viz, it must expose a substantial portion of the channel wall, and it must not interfere appreciably with the electric field passing thru the channel.

By the use of a larger or smaller bag, within the cell frame the volume capacity of the cell may be adjusted within wide limits. For example, in one apparatus where the height and width of the cell frame were 12 and 5 inches respectively, a bag as small as 1 inch wide by 9 inches high was used, giving a volume capacity of 50 ml., while in the same apparatus. a bag 3.5 inches wide by 13 inches high gave a volume capacity of 250 ml. The volume capacity of the cell may also be adjusted by shifting the position of the bag with relation to the frame. In the example cited, when the small bag is shifted toward the top of the frame, in such a way that the bottom of the bag projects 1 cm. into the bottom recess, the volume capacity is reduced to 20 ml.

One of the desirable advantages of the bag and frame construction as described in this specification is that the bag which is to contain the colloid dispersion, may be independently sterilized by the usual bacteriological techniques such as immersion in boiling water. In this way bacterial contamination of the dispersion may be completely prevented. Furthermore, as an additional precaution against contamination, the bag may be discarded after a single use.

The present invention provides means for preventing the remixing of the separated fractions at the conclusion of the fractionation. This means is the occlusion of the lumen of the channel. One form of a device for this occlusion is shown in Fig. 1, where 30 is a wedge valve which is lowered into and completely fills the lumen of the channel when occlusion is desired. Other forms of this means may be used within the teaching of the present specification, such as a device for applying pressure across the walls of the channel from the outside, thus causing the walls to collapse. The preferred point at which the channel is to be occluded is at or near the top thereof where the channel enters and communicates with the top reservoir.

The construction of a laminar channel previously referred to in this specification is also exhibited in Fig. 1, which shows a channel with two. laminae, altho more than two may be advantageously used. Adjacent laminae are separated by a sheet 42 of semipermeable membrane suspended within the bag. The sheet is wider than the channel and approximately the same in vertical dimension as the channel and in the design shown is suspended therein by clamping the edges of the sheet between the two plates of the frame. In this type of construction the average thickness of each lamina may be amout /2 to 5 mm. or even more, as for a single channel. In the construction shown the desired thickness is attained by the bulging of the external walls of the channel while the single dividing membrane is planar. In constructing channels of more than two laminae, having more than one dividing membrane, separation of the membranes may be maintained by spacing elements at the sides of the channel.

Two vertical bags parallel to each other may be clamped between the plates of the external frame, thereby forming two separate cells each with its reservoirs and channels. Even more than two separate cells may be formed in this way, provided only that adequate thickness is maintained in each channel.

To obtainthe maximum efliciency from the apparatus a large electric current, up to 100 or even more milliamperes per square centimeter of channel face area must be passed thru the cell. The large amount of heat generated in the cell by this current interferes seriously with the operation of the apparatus. I have found that this heat may be dissipated by rapidly circulating buffer, first thru a cooling device, and then thru the electrode chambers. The inlets 25, 26 are used to carry the steam of buffer. I have found that for each watt of electrical energy passed thru the cell a buffer flow of about at least one liter per hour is required to maintain the contents of the cell within one degree centigrade of the ambient temperature, and even better results are obtained if the flow is increased over the figure given. For example, in a cell whose channel is 15 cm. high and 2 cm. wide a current of 100 ma. per sq. cm. developed heat at a rate of 60 Watts. When the inflowing buffer was maintained at 4 C. a rate of flow of 60 liters per hour maintained the contents of the cell at 5 C. Without such cooling the temperature of the cell contents rapidly rose to 100 C. and destroyed the colloid dispersion within the cell.

The present invention may take other desirable forms. For example, the bag described above may be stretched over an internal frame which maintains it in the proper shape. I wish it to be understood that my invention is not to be limited to the specific form, size, or arrangement of parts herein shown and described.

I claim:

1. In an apparatus for fractionating colloid dispersions the combination of: a bag of flexible semipermeable membrane capable of retaining colloid dispersions; a frame of electrically non-conducting construction external thereto which holds said bag in a vertical position, said frame compressing center portions of said bag so as to form a narrow vertical channel connecting upper and lower portions of said bag, which upper and lower portions form reservoirs communicating with said channel, said frame exposing aligned opposed portions of said bag forming walls of said channel whereby an electric field may be passed through and maintained within the bag across said channel; whereby a leak-free cell suitable for electrophoresis convection is obtained.

2. In an apparatus for fractionating colloid dispersions the combination of: a continuous flexible tube of semipermeable membrane capable of retaining colloid dispersions; a frame which holds said tube in the shape of a vertical U with both open ends of the tube at the top thereof, in such a way that a continuous body of liquid may be maintained within the lumen of the tube; said frame compressing center portions of the vertical arms of the U-shaped tube so as to form two substantially parallel narrow vertical channels providing means of liquid communication between the lower portion of the tube and upper portions of the tube respectively, said frame exposing aligned opposed portions of said channels in such a way that a horizontal electric field may be passed through said channels in such a way that a leakfree cell suitable for electrophoresis convection is obtained.

3. In an apparatus for fractionating colloid dispersions the combination of: a cell of flexible semipermeable membrane capable of retaining holding said cell, said means including portions external to the cell'which compress center portions of said cell so as to form a substantially vertical narrow channel of liquid commtmication Within said cell between lower po r tions and upper portions of said cell, the positional relation of said cell to said external portions being variable; in such a way that the volume capacity of the cell is adjustable.

4. In an apparatus for fractionating colloid dispersions the combination of: a box made of an electrically nonconducting material; a frame having two juxtaposed plates of electrically non-conducting material having aligned openings therein forming a hole through saidv frame, said plates also having adjoining top and bottom recesses forming top and bottom chambers which cornmunicate with the hole, said frame being positioned within said box and partitioning it into compartments; means of liquid communication between said compartments; a tube of flexible semipermeable membrane capable of retaining colloid dispersions clamped between said 7 plates and folded at the bottom in such a way that upper end portions of the tube are located in the upper chamber and the'fold of said tube is located in the bottom chamber, a portion of said tubes being exposed by the aforementioned hole which is completely covered by said tube, in such a way that a cell is formed having a reser voir at the bottom which communicates with two vertical channels each of which communicates with a reservoir at the top, in which cell a continuous body of liquid can be maintained; a means for occluding simultaneously the lumens of both channels; electrodes positioned within said box, by means of which an electric field may be passed through the cell; means for pumping a liquid contained in said box, the suction of said means communicating with one of the compartments of the box; means for cooling a liquid, through which the aforementioned pumping means may pump liquid and return it to the box, the point of return being a compartment other than the one with which the suction of said pumping means communicates, in such a way that a leakfree apparatus of adjustable volume is obtained.

5. In an apparatus for fractionating colloid dispersions the combination of: a bag of semipermeable membrane capable of retaining colloid dispersions; a frame external thereto which holds said bag in the shape of at least one substantially flat vertical channel, the width and height thereof being materially greater than the thickness thereof and the thickness thereof being from /2 to 5 mm. on the average, said frame further holding other portions of said bag in the shape of reservoirs communicating directly with said channel, one reservoir communicating with the top and another reservoir communicating with the bottom of said channel, the walls of said reservoirs consisting of portions of said bag continuous with the portions of said bag forming said channel whereby a cell of smooth internal contours is obtained; said frame being provided with aligned openings exposing opposed portions of said bag forming the walls of said channel; means for occluding the lumen of said channel, the point of occlusion being located near the junction of said channel and said top reservoir; means for applying and maintaining a horizontal electric field across said channel through said openings; and means.

structed of electrically non-conducting material and being lined with semipermeable membrance continuous with colloid dispersions; means for References Cited in the file of this patent UNITED STATES PATENTS 442,203 Roberts Dec. 9, 1890 10 Kean May 31, 1932 Cardone July 6, 1937 FOREIGN PATENTS Great Britain Jan. 17, 1941 Great Britain Dec. 21, 1939 

1. IN AN APPARATUS FOR FRACTIONATING COLLOID DISPERSIONS THE COMBINATION OF: A BAG OF FLEXIBLE SEMIPERMEABLE MEMBRANE CAPABLE OF RETAINING COLLOID DISPERSIONS; A FRAME OF ELECTRICALLY NON-CONDUCTING CONSTRUCTION EXTERNAL THERETO WHICH HOLDS SAID BAG IN A VERTICAL POSITION, SAID FRAME COMPRESSING CENTER PORTIONS OF SAID BAG SO AS TO FORM A NARROW VERTICAL CHANNEL CONNECTING UPPER AND LOWER PORTIONS OF SAID BAG, WHICH UPPER AND LOWER PORTIONS FORM RESERVOIRS COMMUNICATING WITH SAID CHANNEL, SAID FRAME EXPOSING ALIGNED OPPOSED PORTIONS OF SAID BAG FORMING WALLS OF SAID CHANNEL WHEREBY AN ELECTRIC FIELD MAY BE PASSED THROUGH AND MAINTAINED WITHIN THE BAG ACROSS SAID CHANNEL; WHEREBY A LEAK-FREE CELL SUITABLE FOR ELECTROPHORESIS CONVECTION IS OBTAINED. 